The Selective NLRP3-inflammasome inhibitor MCC950 Mitigates Post-resuscitation Myocardial Dysfunction and Improves Survival in a Rat Model of Cardiac Arrest and Resuscitation.

PURPOSE: To investigate the effects of the selective NLRP3 inflammasome inhibitor MCC950 on post-resuscitation myocardial function and survival in a rat model of cardiopulmonary resuscitation (CPR). METHODS: Thirty-six Sprague Dawley rats were randomized into three groups: (1) MCC950, (2) control, and (3) sham. Each group consisted of a 6 h non-survival subgroup (n = 6) and a 48 h survival subgroup (n = 6). Ventricular fibrillation (VF) was induced and untreated for 6 min. CPR was initiated and continued for 8 min. Resuscitation was attempted with a 4 J defibrillation. MCC950 (10 mg/kg) or vehicle was administered via intraperitoneal injection immediately after the return of spontaneous circulation (ROSC). Myocardial function and sublingual microcirculation were measured after ROSC in the non-survival subgroups. Plasma levels of interleukin Iß (IL-1ß) and cardiac troponin I (cTnI) were measured at baseline and 6 h in the non-survival subgroups. Heart tissue was harvested to measure the NLRP3 inflammasome constituents, including NLRP3, apoptosis-associated speck-like protein (ASC), Caspase-1, and IL-1ß. Survival duration and neurologic deficit score (NDS) were recorded and evaluated among survival groups. RESULTS: Post-resuscitation myocardial function and sublingual microcirculation were improved in MCC950 compared with control (p < 0.05). IL-1ß and cTnI were decreased in MCC950 compared to control (p < 0.01). The MCC950 treated groups showed significantly reduced ASC, caspase-1, and IL-1ß compared with the control group (p < 0.05). Survival at 48 h after ROSC was greater in MCC950 (p < 0.05) with improved NDS (p < 0.05). CONCLUSION: Administration of MCC950 following ROSC mitigates post-resuscitation myocardial dysfunction and improves survival.

Abnormal expression and clinical significance of surface receptors on natural killer cells in the peripheral blood of patients with non-small cell lung cancer.

Natural killer (NK) cells typically function as frontline lymphocytes against cancer although little is known about their engagement in non-small cell lung cancer (NSCLC). This study compared the performance and activity of NK cells and their subsets in the peripheral blood of NSCLC sufferers and healthy participants. In total, 67 healthy controls (40 males; 59.7%) and 56 patients with NSCLC (35 males; 62.5%) were included (mean age, 66.6 years). Flow cytometry identified NK cells and their subpopulations in external blood, and the total number, proportion, activity, surface activating, and inhibitory receptor expression levels were determined. Results showed that NK cell surface receptors CD107a, IFN-γ, and TNF-α activity were markedly reduced in lung cancer patients compared to healthy controls. The number and ratio of NK cells within the lymphocyte population were decreased in patients. The concentration of the inhibitory receptors TIGIT, TIM-3, CD96, PD-1, and Siglec-7 were increased in patients, whereas the expression level of the activating receptor NKP30 was decreased. Moreover, the expression levels of IFN-γ, TIGIT, CD96, PD-1, and TIM-3 were correlated with the clinical phase of NSCLC. These findings suggest that surface receptors from NK cells are likely to be involved in the evolution of NSCLC.

Synergistic Effect of Notch-3-Specific Inhibition and Paclitaxel in Non-Small Cell Lung Cancer (NSCLC) Cells Via Activation of The Intrinsic Apoptosis Pathway.

BACKGROUND Lung cancers are resistant to conventional chemotherapeutic interventions such as paclitaxel. Notch signaling is crucial in the chemoresistance of lung cancer cells. The Notch inhibitor gamma-secretase inhibitor (GSI) inhibits the Notch signaling pathway. MATERIAL AND METHODS Here, we evaluated how Notch-3 inhibition by GSI can enhance the sensitivity of lung cancer cells to paclitaxel. To study how Notch-3-specific inhibition affects non-small cell lung cancer (NSCLC), we compared the cell viability, apoptosis, and colony formation of A549 and H1299 cells treated with Notch-3 siRNA and GSI. RESULTS The expression levels of Notch-3 or Notch intracellular domain 3 (NICD3) and apoptosis-related proteins were measured and compared between different groups. Notch-3 was significantly overexpressed in both cell lines, and Notch-3 expression was elevated after paclitaxel treatment, indicating activation of the Notch signaling pathway. Inhibition of the Notch signaling pathway by GSI and Notch-3 siRNA reduced cell proliferation and induced apoptosis in A549 and H1299 cells, thereby boosting sensitivity of the cell lines to paclitaxel. Concomitant treatment with paclitaxel and GSI or siRNA downregulated Bcl-2 expression and upregulated Bax expression levels. CONCLUSIONS These results indicate a synergistic effect of Notch-3-specific inhibition and paclitaxel through alteration of the intrinsic apoptosis pathway, which was involved in Notch-3-induced chemoresistance in NSCLC cells, and GSI inhibited Notch-3-induced chemoresistance in a concentration-dependent manner. This approach that combines Notch-3-specific inhibition and paclitaxel would be likely to apply in NSCLC.

JAK activity regulates mesoderm cell fate by controlling MESP1 expression.

Cardiac development requires precise gene expression programs at each developmental stage guided by multiple signaling pathways and transcription factors (TFs). MESP1 is transiently expressed in mesoderm, and is essential for subsequent cardiac development, while the precise mechanism regulating its own transcription and mesoderm cell fate is not fully understood. Therefore, we developed a high content screen assay to identify regulators of MESP1 expression in mesodermal cells differentiated from human pluripotent stem cells (hPSCs). The screen identified CYT387, a JAK1/JAK2 kinase inhibitor, as a potent activator of MESP1 expression, which was also found to promote cardiomyocyte differentiation in vitro. Mechanistic studies found that JAK inhibition promotes MESP1 expression by reducing cytoplasmic calcium concentration and subsequently activating canonical WNT signaling. Our study identified a role of JAK signaling in early mesodermal cells, and sheds light on the connection between the JAK-STAT pathway and transcriptional regulation of MESP1, which expands our understanding of mesoderm and cardiac development.

Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration.

Conventional type 1 dendritic cells (cDC1) are the essential antigen-presenting DC subset in antitumor immunity. Suppressing B-cell lymphoma 9 and B-cell lymphoma 9-like (BCL9/BCL9L) inhibits tumor growth and boosts immune responses against cancer. However, whether oncogenic BCL9/BCL9L impairs antigen presentation in tumors is still not completely understood. Here, we show that targeting BCL9/BCL9L enhanced antigen presentation by stimulating cDC1 activation and infiltration into tumor. Pharmacological inhibition of BCL9/BCL9L with a novel inhibitor hsBCL9z96 or Bcl9/Bcl9l knockout mice markedly delayed tumor growth and promoted antitumor CD8+ T cell responses. Mechanistically, targeting BCL9/BCL9L promoted antigen presentation in tumors. This is due to the increase of cDC1 activation and tumor infiltration by the XCL1-XCR1 axis. Importantly, using single-cell transcriptomics analysis, we found that Bcl9/Bcl9l deficient cDC1 were superior to wild-type (WT) cDC1 at activation and antigen presentation via NF-κB/IRF1 signaling. Together, we demonstrate that targeting BCL9/BCL9L plays a crucial role in cDC1-modulated antigen presentation of tumor-derived antigens, as well as CD8+ T cell activation and tumor infiltration. Targeting BCL9/BCL9L to regulate cDC1 function and directly orchestrate a positive feedback loop necessary for optimal antitumor immunity could serve as a potential strategy to counter immune suppression and enhance cancer immunotherapy.

Necrosulfonamide improves post-resuscitation myocardial dysfunction via inhibiting pyroptosis and necroptosis in a rat model of cardiac arrest.

The systemic inflammatory response following global myocardial ischemia/reperfusion (I/R) injury is a critical driver of poor outcomes. Both pyroptosis and necroptosis are involved in the systemic inflammatory response and contribute to regional myocardial I/R injury. This study aimed to explore the effect of necrosulfonamide (NSA) on post-resuscitation myocardial dysfunction in a rat model of cardiac arrest. Sprague-Dawley rats were randomly categorized to Sham, CPR and CPR-NSA groups. For rats in the latter two groups, ventricular fibrillation was induced without treatment for 6 min, with cardiopulmonary resuscitation (CPR) being sustained for 8 min. Rats were injected with NSA (10 mg/kg in DMSO) or vehicle at 5 min following return of spontaneous circulation. Myocardial function was measured by echocardiography, survival and neurological deficit score (NDS) were recorded at 24, 48, and 72 h after ROSC. Western blotting was used to assess pyroptosis- and necroptosis-related protein expression. ELISAs were used to measure levels of inflammatory cytokine. Rats in the CPR-NSA group were found to exhibit superior post-resuscitation myocardial function, and better NDS values in the group of CPR-NSA. Rats in the group of CPR-NSA exhibited median survival duration of 68 ± 8 h as compared to 34 ± 21 h in the CPR group. After treatment with NSA, NOD-like receptor 3 (NLRP3), GSDMD-N, phosphorylated-MLKL, and phosphorylated-RIP3 levels in cardiac tissue were reduced with corresponding reductions in inflammatory cytokine levels. Administration of NSA significantly improved myocardial dysfunction succeeding global myocardial I/R injury and enhanced survival outcomes through protective mechanisms potentially related to inhibition of pyroptosis and necroptosis pathways.

Discovery of a novel, potent and selective small-molecule inhibitor of PD-1/PD-L1 interaction with robust in vivo anti-tumour efficacy.

BACKGROUND AND PURPOSE: PD-1/PD-L1 antibodies have achieved great success in clinical treatment. However, monoclonal antibody drugs also have challenges, such as high manufacturing costs, poor diffusion, low oral bioavailability and limited penetration into tumour tissue. The development of small-molecule inhibitors of PD-1/PD-L1 interaction represents a promising perspective to overcome the above challenges in cancer immunotherapy. EXPERIMENTAL APPROACH: We explored structural activity relationships and used biochemical assays to generate a lead compound (ZE132). CD8+ T-cells killing assay and Ifng expression assay were used to verify the in vitro cellular activity of ZE132. Off-target study was performed to verify the selectivity. Syngeneic mouse models were used to verify the in vivo activity of ZE132 in tumour immune microenvironment (TIME). We also performed pharmacokinetics profiling in mice and The Cancer Genome Atlas database analysis. KEY RESULTS: ZE132 can effectively inhibit the PD-1/PD-L1 interactions in vitro, and it has a potent affinity to PD-L1. ZE132 shows robust anti-tumour effects in vivo, better than anti-PD-1 antibody. In the analysis of TIME, we found that ZE132 treatment promotes cytotoxic T-cell tumour infiltration and induces IL-2 expression. In addition, ZE132 elicits strong inhibitory effects on the mRNA expression of TGF-ß, which may serve as a potential biomarker to predict responsiveness to PD-1/PD-L1 immunotherapies. CONCLUSION AND IMPLICATIONS: We identified a new lead compound ZE132 targeting PD-1/PD-L1 interactions, not only showing favourable drug-like properties in vitro and in vivo but also showing the advantage of overcoming the barrier of TIME compared to anti-PD-1 antibody.

Cerebral and myocardial mitochondrial injury differ in a rat model of cardiac arrest and cardiopulmonary resuscitation.

Brain mitochondria are more sensitive to global ischemia compared to heart mitochondria. Complex I in the electron transport chain (ETC) is sensitive to ischemic injury and is a major control point of the rate of ADP stimulated oxygen consumption. The purpose of this study was to explore whether changes in cerebral and myocardial mitochondria differ after cardiac arrest. Animals were randomized into 4 groups (n = 6): 1) Sham 2) VF 3) VF+CPR 4) ROSC 1hr. Ventricular Fibrillation (VF) was induced through a guide wire advanced from the right jugular vein into the ventricle and untreated for 8 min. Resuscitation was attempted with a 4J defibrillation after 8 min of cardiopulmonary resuscitation (CPR). Brain mitochondria and cardiac mitochondrial subpopulations were isolated. Calcium retention capacity was measured to assess susceptibility to mitochondrial permeability transition pore opening. ADP stimulated oxygen consumption and ETC activity assays were performed. Brain mitochondria are far more sensitive to injury during cardiac arrest and resuscitation compared to cardiac mitochondria. Complex I is highly sensitive to injury in brain mitochondria. With markedly decreased calcium retention capacity, mitochondria contribute to cerebral reperfusion injury. Therapeutic preservation of cerebral mitochondrial activity and mitochondrial function during cardiac arrest may improve post-resuscitation neurologic function.

Effects of NLRP3 inflammasome blockade on postresuscitation cerebral function in a rat model of cardiopulmonary resuscitation.

Cardiac arrest (CA) remains a major public health issue. Inflammatory responses with overproduction of interleukin-1ß regulated by NLRP3 inflammasome activation play a crucial role in cerebral ischemia/reperfusion injury. We investigated the effects of the selective NLRP3-inflammasome inhibitor MCC950 on post-resuscitation cerebral function and neurologic outcome in a rat model of cardiac arrest. Thirty-six male rats were randomized into the MCC950 group, the control group, or the sham group (N = 12 of each group). Each group was divided into a 6 h non-survival subgroup (N = 6) and a 24 h survival subgroup (N = 6). Ventricular fibrillation (VF) was electrically induced and untreated for 6 min, followed by 8 min of precordial compressions and mechanical ventilation. Resuscitation was attempted with a 4J defibrillation. Either MCC950 (10 mg/kg) or vehicle was injected intraperitoneally immediately after the return of spontaneous circulation (ROSC). Rats in the sham group underwent the same surgical procedures without VF and CPR. Brain edema, cerebral microcirculation, plasma interleukin Iß (IL-1ß), and neuron-specific enolase (NSE) concentration were measured at 6 h post-ROSC of non-survival subgroups, while 24 h survival rate, neurological deficits were measured at 24 h post-ROSC of survival subgroups. Post-resuscitation brain edema was significantly reduced in animals treated with MCC950 (p < 0.05). Cerebral perfused vessel density (PVD) and microcirculatory flow index (MFI) values were significantly higher in the MCC950 group compared with the control group (p < 0.05). The plasma concentrations of IL-1ß and NSE were significantly decreased in animals treated with MCC950 compared with the control group (p < 0.05). 24 h-survival rate and neurological deficits score (NDS) was also significantly improved in the MCC950 group compared with the control group (p < 0.05). NLRP3 inflammasome blockade with MCC950 at ROSC reduces the circulatory level of IL-1ß, preserves cerebral microcirculation, mitigates cerebral edema, improves the 24 h-survival rate, and neurological deficits.

BCL9 regulates CD226 and CD96 checkpoints in CD8+ T cells to improve PD-1 response in cancer.

To date, the overall response rate of PD-1 blockade remains unsatisfactory, partially due to limited understanding of tumor immune microenvironment (TIME). B-cell lymphoma 9 (BCL9), a key transcription co-activator of the Wnt pathway, is highly expressed in cancers. By genetic depletion and pharmacological inhibition of BCL9 in tumors, we found that BCL9 suppression reduced tumor growth, promoted CD8+ T cell tumor infiltration, and enhanced response to anti-PD-1 treatment in mouse colon cancer models. To determine the underlying mechanism of BCL9's role in TIME regulation, single-cell RNA-seq was applied to reveal cellular landscape and transcription differences in the tumor immune microenvironment upon BCL9 inhibition. CD155-CD226 and CD155-CD96 checkpoints play key roles in cancer cell/CD8+ T cell interaction. BCL9 suppression induces phosphorylation of VAV1 in CD8+ T cells and increases GLI1 and PATCH expression to promote CD155 expression in cancer cells. In The Cancer Genome Atlas database analysis, we found that BCL9 expression is positively associated with CD155 and negatively associated with CD226 expression. BCL9 is also linked to adenomatous polyposis coli (APC) mutation involved in patient survival following anti-PD-1 treatment. This study points to cellular diversity within the tumor immune microenvironment affected by BCL9 inhibition and provides new insights into the role of BCL9 in regulating CD226 and CD96 checkpoints.

N-acetylcysteine alleviates post-resuscitation myocardial dysfunction and improves survival outcomes via partly inhibiting NLRP3 inflammasome induced-pyroptosis.

BACKGROUND: NOD-like receptor 3 (NLRP3) inflammasome is necessary to initiate acute sterile inflammation. Increasing evidence indicates the activation of NLRP3 inflammasome induced pyroptosis is closely related to reactive oxygen species (ROS) in the sterile inflammatory response triggered by ischemia/reperfusion (I/R) injury. N-acetylcysteine (NAC) is an antioxidant and plays a protective role in local myocardial I/R injury, while its effect on post-resuscitation myocardial dysfunction, as well as its mechanisms, remain elusive. In this study, we aimed to investigate the effect of NAC on post-resuscitation myocardial dysfunction in a cardiac arrest rat model, and whether its underlying mechanism may be linked to ROS and NLRP3 inflammasome-induced pyroptosis. METHODS: The rats were randomized into three groups: (1) sham group, (2) cardiopulmonary resuscitation (CPR) group, and (3) CPR + NAC group. CPR group and CPR + NAC group went through the induction of ventricular fibrillation (VF) and resuscitation. After return of spontaneous circulation (ROSC), rats in the CPR and CPR + NAC groups were again randomly divided into two subgroups, ROSC 6 h and ROSC 72 h, for further analysis. Hemodynamic measurements and myocardial function were measured by echocardiography, and western blot was used to detect the expression of proteins. RESULTS: Results showed that after treatment with NAC, there was significantly better myocardial function and survival duration; protein expression levels of NLRP3, adaptor apoptosis-associated speck-like protein (ASC), Cleaved-Caspase-1 and gasdermin D (GSDMD) in myocardial tissues were significantly decreased; and inflammatory cytokines levels were reduced. The marker of oxidative stress malondialdehyde (MDA) decreased and superoxide dismutase (SOD) increased with NAC treatment. CONCLUSIONS: NAC improved myocardial dysfunction and prolonged animal survival duration in a rat model of cardiac arrest. Moreover, possibly by partly inhibiting ROS-mediated NLRP3 inflammasome-induced pryoptosis.

Effects of Polyethylene Glycol-20k on Coronary Perfusion Pressure and Postresuscitation Myocardial and Cerebral Function in a Rat Model of Cardiac Arrest.

Background Epinephrine increases the rate of return of spontaneous circulation. However, it increases severity of postresuscitation myocardial and cerebral dysfunction and reduces duration of survival. We investigated the effects of aortic infused polyethylene glycol, 20 000 molecular weight (PEG-20k) during cardiopulmonary resuscitation on coronary perfusion pressure, postresuscitation myocardial and cerebral function, and duration of survival in a rat model of cardiac arrest. Methods and Results Twenty-four male rats were randomized into 4 groups: (1) PEG-20k, (2) epinephrine, (3) saline control-intravenous, and (4) saline control-intra-aortic. Cardiopulmonary resuscitation was initiated after 6 minutes of untreated ventricular fibrillation. In PEG-20k and Saline-A, either PEG-20k (10% weight/volume in 10% estimated blood volume infused over 3 minutes) or saline was administered intra-aortically after 4 minutes of precordial compression. In epinephrine and placebo groups, either epinephrine (20 µg/kg) or saline placebo was administered intravenously after 4 minutes of precordial compression. Resuscitation was attempted after 8 minutes of cardiopulmonary resuscitation. Sublingual microcirculation was measured at baseline and 1, 3, and 5 hours after return of spontaneous circulation. Myocardial function was measured at baseline and 2, 4, and 6 hours after return of spontaneous circulation. Neurologic deficit scores were recorded at 24, 48, and 72 hours after return of spontaneous circulation. Aortic infusion of PEG-20k increased coronary perfusion pressure to the same extent as epinephrine. Postresuscitation sublingual microcirculation, myocardial and cerebral function, and duration of survival were improved in PEG-20k (P<0.05) compared with epinephrine (P<0.05). Conclusions Aortic infusion of PEG-20k during cardiopulmonary resuscitation increases coronary perfusion pressure to the same extent as epinephrine, improves postresuscitation myocardial and cerebral function, and increases duration of survival in a rat model of cardiac arrest.

Increased expression of Toll-like receptors 4 and 9 in human lung cancer.

BACKGROUND: It has been reported lately that Toll-like receptors (TLRs) play an essential role in the activation of innate immunity, and TLRs are expressed in a large number of immune cells like B-lymphocytes, monocytes, plasmacytoid dendritic cells and at low levels in human respiratory cells as well as epithelial cells. In the present study, we investigated whether there is a relationship between the expression of TLR4 or TLR9 and the clinical or pathological changes in human lung cancer. METHOD: Protein expression of TLR4 and TLR9 was assessed by using immunohistochemistry and western blotting. mRNA expressions of TLR4 and TLR9 were detected by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: High TLR4 and TLR9 mRNA signal intensity was found in the majority of lung cancer specimens. In contrast, tumor-free lung tissue showed lower signal intensity. Consistently, the low amount of TLR4 and TLR9 protein expression was found in tumor-free lung tissue, while they were strongly expressed in lung cancer tissue. In addition, we found for the first time that the differentiation degree of tumor cells was positively correlated with the expression level of TLR4. There was no relationship between the expressions of TLR4 or TLR9 and patients' age, gender, smoking, the histological type of tumor, lymph node metastasis, and tumor node metastases (TNM) stage. CONCLUSIONS: We found that both mRNA and protein levels of TLR4 and TLR9 were strongly expressed in lung cancer tissue. In addition, we reported for the first time a positive correlation between the expression level of TLR4 and malignancy of lung cancer. These results suggested that TLR4 and TLR9 may be involved in the development of lung cancer which may have the potentials for the treatment of this malignant tumor.

[Mechanism of regulation of leptin expression in human lung adenocarcinoma cells by hypoxia inducible factor-1alpha: a preliminary study].

OBJECTIVE: To investigate the regulation of leptin expression in human lung adenocarcinoma cells by hypoxia inducible factor-1 (HIF-1alpha) and the mechanism thereof. METHODS: Lung adenocarcinoma tissues were collected from 42 patients during operation and samples of corresponding adjacent lung tissue were obtained from 16 of the 42 specimens. The expression levels of HIF-1alpha and leptin were detected by immunohistochemical staining. Human lung adenocarcinoma cells of the line A549 cells were cultured for 0, 12, 24, and 48 h respectively, exposed to hypoxia induced by CoCl2. Other A549 cells were treated with GL331, a kind of HIF-1alpha inhibitor, of the concentrations of 0, 5, 10, or 20 micromol/L under normoxic condition for 24 h, and then were exposed to hypoxia induced by CoCl2 for 24 h. RT-PCR and Western-blotting were used to examine the mRNA and protein expression levels of HIF-1alpha and leptin in different groups. RESULTS: Immunohistochemistry showed that the positive rates of HIF-1alpha and leptin in the lung adenocarcinoma tissue were 57.1% and 69.0% respectively, both significantly higher than those in the adjacent normal lung tissues (18.8% and 25.0% respectively, both P<0.01). The protein expression levels of HIF-1alpha in the hypoxia 0, 12, 24, and 48 h groups were 0.314+/-0.030, 0.552+/-0.027, 0.743+/-0.015, and 0.799+/-0.010 respectively, and the protein expression levels of leptin were 0.398+/-0.016, 0.633+/-0.036, 0.796+/-0.008, and 0.942+/-0.088 respectively, increasing in a time dependent manner too. The mRNA expression levels of leptin in the 4 hypoxia groups were 0.144+/-0.009, 0.336+/-0.017, 0.524+/-0.013, and 0.671+/-0.021 respectively, increasing in a time dependent manner, however, there was no significant differences in the mRNA expression levels of HIF-1alpha among the groups exposed to hypoxia for different courses of time (all P>0.05). The protein expression levels of HIF-1alpha and leptin, and the mRNA expression levels of leptin in the groups exposed to hypoxia for different courses of time were all significantly higher than those of the control (0 h hypoxia) group (all P<0.01). The mRNA and protein expression levels of leptin in the A549 cells exposed to GL331 and hypoxia were decreased dose-dependently. CONCLUSION: The expression of leptin is up-regulated by hypoxia and regulated by HIF-1alpha.